Summary of Work: Our research efforts encompassed two general areas: (1) The modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins, and (2) the instrumental development and applications of (a) vibrational Raman and infrared spectroscopic imaging techniques and (b) the clinical utilization of visible-reflectance hyperspectral imaging approaches. (1) Our interest in characterizing the sizes and formation of fluctuating lipid microdomains within biomembranes, using vibrational infrared and Raman spectroscopic techniques, were focused on lipid cluster formation and their modulatory influence on induced conformational changes occurring within integral membrane proteins. Bacteriorhodopsin was used as a model system to demonstrate the effects from the lateral compressibility properties of these quantified lipid microaggregates on the motional characteristics of the protein?s transmembrane helices. In studying spectroscopically specific lipid chain order/disorder effects within the microdomains, appropriate acyl chain deuteration allowed the vibrational dynamics of each chain moiety to be monitored separately. Additionally, using infrared spectroscopic techniques, we examined in detail the lipid control of both the photocycle activity of the M-state intermediates of bacteriorhodopsin and the conformational flexibility of the protein's integral membrane alpha helices. (2A) Considerable emphasis was placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan interferometry with state-of-the-art infrared sensitive two-dimensional focal plane array detectors. The integration of high performance digital imaging with noninvasive, high resolution optical spectroscopy allows a visualization of the spatial distribution of distinct chemical species in a variety of host environments. The power of the technique is also manifest in the simultaneous acquisition of an infrared spectrum for each spatial location. As one example of the utility of the infrared imaging technique in diagnostic pathology, we applied the technique to both supervised and unsupervised prostate histopathology involving large numbers of tissue sections in the form of tissue microarrays. This spectroscopic method eliminates the necessity for chemically stained tissue. Further, the high throughput approach inherent in the use of tissue microarrays allows the efficient and effective acquisition of the vibrational spectral signatures for the pathologic analyses of biopsied representations of control specimens, prostatic intraepithelial neoplastic samples, samples manifesting benign prostatic hyperplasia and adenocarcinoma tissues. In this case, our imaging instrumentation incorporated highly sensitive linear array detection for rapidly recording hypercube spectral data. For spectroscopically elucidating the various histologic features present in prostate tissue, extraordinarily large spectral training sets and appropriate spectroscopic metrics were developed for distinguishing ten morphological entities occurring in prostatic tissue. As examples, both the use of the Maximum Gaussian Likelihood Method and, separately, a probabilistic classification model allowed the objective, automated delineation of the ten histologic categories to the order of 95%. With regard to our general infrared imaging instrumentation, a number of enhancing features were made in the optics, in detector configurations, and in data collection paradigms. In particular, we have implemented and utilized a generalized form of interferometric rapid-, or continuous, scan infrared spectroscopic imaging (to be distinguished from interferometer step-scan approaches) for utilization with any type of focal plane array detector to image nonreversible dynamic events in the order of seconds. Further, we are the first group to implement time-resolved Fourier-transform infrared spectroscopic imaging which permits, for example, the visualization of repetitive dynamic processes with half lives on the order of tenths of milliseconds. The examples used in this case involved specific polymer liquid crystal composites. (2B) Our imaging approaches have also been extended to the visible spectral region in which reflectance spectra are obtained using CCD detection and appropriate liquid crystal tunable filters for wavelength discrimination. This particular noninvasive reflectance unit has been used successfully in clinical venues for imaging hemoglobin tissue oxygenation saturation in sickle cell disease patient in which we examined the effects of nitric oxide (NO) stimulation, inhibition and administration. In addition to the effects of NO gas, actetylcholine, an agonist that stimulates the release of NO from the endothelium, and sodium nitroprusside, a direct NO donor and endothelium-independent vasodilator were examined. We concluded that patients with sickle cell disease exhibit impaired tissue oxygenation despite having resting blood flow values that were two-fold higher than healthy African American subjects. Furthermore, when blood flow was pharmacologically increased by seven-fold, tissue oxygenation was improved, but remained well below healthy subjects. This versatile, visible reflectance imaging approach provides a useful, real time probe of patients with vascular disease and allows a novel means for assessing disease severity and disease progression.